The present invention resides in the field of genetic analysis.
Cytogenetic testing is still in its infancy. Current cytogenetic methods are limited to analysis of gene aberrations easily detectable in cells, nuclei, or chromosomes, in part, because slide based methods are highly manual. Analysis of aberrations present in low frequency is not routinely performed in the clinical setting because many slides of cells, nuclei or chromosomes would have to be evaluated to establish statistical frequency.
Banding is the classical approach used for analyzing chromosomes in metaphase spreads. This method is based on staining which results in dark bands in the region of the chromosome where the chromatin occurs at higher density. The banding pattern is specific for each chromosome and allows identification for karyotyping, which is the determination of each chromosome""s copy number. However, banding resolution is not sufficient to detect small deletions or additions of chromosomal mass, which occur in a variety of disease conditions, particularly in cancers.
Fluorescent in situ hybridization is another approach used to localize genomic DNA fragments or to paint whole chromosomes and to detect and characterize genetic abnormalities including translocations (31, 40), aneusomy (41, 42), and gene amplification (43). These genetic abnormalities can be detected in individual cells, chromosomes, or nuclei to assess of tumor genotype, analyze genetic heterogeneity, and detect malignant cells. To preserve integrity in FISH applications, chromosomes are typically adsorbed onto glass slides for analysis. Analysis therefore requires microscopic evaluation of individual slides limiting automation and rapid sample processing. Fluorescent in situ hybridizations prepared on glass slides rely not only on the assay and reagents but on the instrumentation and the expertise and ingenuity of the scientists using it resulting in poor reproducibility. An inherent limitation to this technology is that at least 100 kb of DNA sequence in a single cell must be present for detection (68-70). In addition, harsh conditions for fixing either tissue or intact cells to a glass slide are less than optimal: up to 90% of the assay sample can be lost from the glass support.
Some chromosomes can also be resolved by fluorescent staining followed by flow cytometry (14,15). Successful chromosome sorting is, however, dependent on the binding characteristics of fluorescent dyes and the extent to which the chromosome of interest can be distinguished from chromosomes of similar size, clumps of chromosomes, and debris containing DNA (13). Although this approach has resulted in the construction of yeast artificial chromosome (YAC) libraries for mapping studies (16) in species which have chromosomes of similar size, such as mouse, arabidopsis, and 20% of the human chromosomes, unambiguous resolution has not been possible. Flow sorting based on dye uptake is possible for well resolved chromosomes, but this method works poorly for chromosomes which are similar in size and base composition, mainly human chromosomes 9-12 and the majority of mouse chromosomes. Furthermore, flow cytometry cannot currently be used to analyze hybridized chromosomes prepared by conventional methods because unfixed chromosomes fall apart using high temperatures and/or formamide.
The invention provides methods of nucleic acid analysis. Such methods entail forming a population of gel microdrops encapsulating a population of biological entities, each entity comprising a nucleic acid, whereby at least some microdrops in the population each encapsulate a single entity. Nucleic acids can be DNA or RNA. The population of gel microdrops is then contacted with a probe under conditions whereby the probe specifically hybridizes to at least one complementary sequence in the nucleic acid in at least one gel microdrop. At least one gel microdrop is then analyzed or detected. The biological entities can be cells, viruses, nuclei and chromosomes.
In some methods, at least 10,000 biological entities are encapsulated. In some methods, the biological entities are not fixed chemically before the contacting step. In some methods, nucleic acids are amplified before the contacting step. Suitable materials for forming droplets include agarose, alginate, carrageenan, or polyacrylamide.
In some methods, nucleic acids are recovered from microdrops by digestion with agarase. Optionally, the recovered DNA can be digested with a restriction enzyme with or without prior digestion of agarase. In some methods, the gel matrix is crosslinked with itself and/or nucleic acid being analyzed, typically, between the denaturation and contacting steps. In some method, the hybridization is performed at a temperature of over 68xc2x0 C. or in the presence of a formamide concentration greater than 20%. In some methods, the microdrops further comprise a reagent that amplifies a signal from the labelled probe. For example, the probe can be labelled with an enzyme, and the reagent can be a substrate for the enzyme.
In some methods, microdrops are isolated by FACS(trademark). In some methods, the biological entities are a population of chromosomes obtained from a population of different cells in a patient. In some methods, the ratio of a subpopulation of microdrops containing a chromosome hybridized to the probe to a subpopulation of microdrops containing a chromosome not hybridized to the probe is determined. In some methods, the probe hybridizes to a nucleic acid segment bearing a mutation and the ratio indicates the proportion of cells in the population bearing the mutation. Such methods are particularly useful for analyzing somatic mutations.
In some methods, an isolated microdrop containing a single chromosome is used to prepare a single chromosomal fragment library. Such a library can in turn be used for preparing probes for a single chromosome, such as painting or reverse painting probes.
Gel microdrops encapsulated biological entities can be stored before or after the hybridization step for a period of at least six months.
The invention further provides methods of diagnosing a disease due to a genetic mutation. Such methods entail obtaining a sample of cells from a patient. A population of chromosomes from the sample in then encapsulated in a population of microdrops. One then contacts the microdrops with a first probe that is complementary to a nucleic acid segment containing the somatic mutation, and a second probe complementary to the chromosome in which the somatic mutation occurs at a site distal to the somatic mutation. The first probe hybridizes to microdrops bearing the chromosome with a somatic mutation and the second probe hybridizes to microdrops bearing the chromosome irrespective whether the somatic mutation is present. One then determines the ratio of microdrops hybridizing to the first probe and hybridizing to the second probe. The ration can then be used to diagnose the existence or prognosis of the disease from the ratio. Such methods are particular useful for diagnosing existence or prognosis of cancer.
The invention further provides methods of chromosome analysis. Such methods entail forming a population of gel microdrops encapsulating a population of nucleic, whereby at least some microdrops in the population each encapsulate a single nucleus. One then contacts the population of gel microdrops with a probe under conditions whereby the probe specifically hybridizes to at least one complementary sequence in at least one chromosome in a nucleus of least one gel microdrop. One then isolates or detects the at least one gel microdrop.
The invention further provides methods of isolating chromosomes. Some such methods entail culturing a population of cells in genistein and colcemid to synchronize chromosomes in metaphase, and isolating chromosomes from the cells. Other methods, which can be used in conjunction or independently of the previously described methods, entail lysing a population of cells to form a lysate. The lysate is then treated with an antibody linked to a magnetic particles, wherein the antibody specifically binds to one or more chromosomes in the cells. Magnetic particles are then isolated from the lysate.
The invention further provides methods of chromosome analysis. Such methods entail forming a population of gel micropdrops encapsulating a population of cells or nuclei, whereby at least some microdrops in the population each encapsulate a single nucleus. One then contacts the population of gel microdrops with a probe under conditions whereby the probe specifically hybridizes to at least one complementary sequence in at least one nucleus in at least one gel microdrop. One then isolates or detects the at least one gel microdrop.
The invention further provides a kit comprising high melting temperature agarose, emulsification equipment, and a label indicating how to use the kit for probe hybridization analysis. Optionally, the kit also includes at least one probe that hybridizes to a nucleic acid.